JGV Papers in Press. Published January 7, 2015 as doi:10.1099/vir.0.000042

Journal of General Virology Characterization of monoclonal antibodies to chicken anemia virus and epitope mapping on its viral protein, VP1 --Manuscript Draft-Manuscript Number:

JGV-D-14-00081R1

Full Title:

Characterization of monoclonal antibodies to chicken anemia virus and epitope mapping on its viral protein, VP1

Short Title:

Neutralizing epitope mapping on VP1 of CAV

Article Type:

Standard

Section/Category:

Animal - Small DNA Viruses

Corresponding Author:

Kunitoshi Imai Obihiro University of Agriculture and Veterinary Medicine Obihiro, JAPAN

First Author:

Dai Q Trinh

Order of Authors:

Dai Q Trinh Haruko Ogawa Vuong N Bui Tugsbaatar Baatartsogt Mugimba K Kizito Shigeo Yamaguchi Kunitoshi Imai

Abstract:

Three (MoCAV/F2, MoCAV/F8, MoCAV/F11) of 4 mouse monoclonal antibodies (mAbs) established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at approximately 50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50-kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize 5 of the strains, indicating distinct antigenic variation among the strains. In blocking immunofluorescence tests with the A2/76infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the antigens and vice versa, suggesting that the 2 mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T), and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least 2 different neutralizing epitopes. However, MoCAV/F8 did not react to EsCAV/F2 or to EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/F2, with a titer of 1:12,800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titers of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.

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Title: Characterization of monoclonal antibodies to chicken anemia virus

and epitope mapping on its viral protein, VP1 Authors: Dai Q. Trinh1,2, Haruko Ogawa1, Vuong N. Bui1,2, Tugsbaatar Baatartsogt1, Mugimba K. Kizito1,3, Shigeo Yamaguchi4, Kunitoshi Imai 1* Affiliations: 1 Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan 2 National Institute of Veterinary Research, 86 Truong Chinh, DongDa, Hanoi, Vietnam 3 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda. 4 Japan Livestock Technology Association, 3-20-9 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan

*Corresponding author: Dr. Kunitoshi Imai.

18 19

Address: Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan

20 21 22 23

Tel: +81-155-49-5892; Fax: +81-155-49-5892; E-mail address: [email protected]

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Summary: 249 words

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Main text: 5,498 words

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Number of table: 2 Tables

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Number of figure: 4 Figures

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Running title: Neutralizing epitope mapping on VP1 of CAV

30

Contents category: Animal viruses – Small DNA

31 32 33 34 35 36 37 38 39 40

The GenBank accession numbers for VP1 gene sequences of CAV strains and VP1, VP2, VP3 gene sequences of escape mutants obtained in this study are as follows: CAV/Japan/G5/79: KJ004667, CAV/Japan/HY/80: KJ004668, CAV/Japan/NI/77: KJ004669, CAV/Japan/HK1/13: KJ126838, CAV/Japan/G1/74: KM226339, CAV/Japan/KY/80: KM226341, CAV/Japan/AO/77: KM226338, CAV/Japan/A1/76: KM226337, CAV/Japan/G7/91: KM226342, CAV/Japan/IBA/94: KM226340, CAV/Japan/NI/92: KM226343, EsCAV/F2: KJ004670 (VP1) and KM235956 (VP2 and VP3), EsCAV/F8: KJ004671 (VP1) and KM235957 (VP2 and VP3), EsCAV/F11: KJ004672 (VP1) and KM235958 (VP2 and VP3).

1

41

Summary

42

Three (MoCAV/F2, MoCAV/F8, MoCAV/F11) of 4 mouse monoclonal antibodies

43

(mAbs) established against the A2/76 strain of chicken anemia virus (CAV) showed

44

neutralization activity. Immunoprecipitation showed a band at approximately 50 kDa in

45

A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50-kDa capsid protein

46

(VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells.

47

MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did

48

not neutralize 5 of the strains, indicating distinct antigenic variation among the strains. In

49

blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11

50

was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the

51

antigens and vice versa, suggesting that the 2 mAbs recognized the same epitope. However,

52

mutations were found in different parts of VP1 of the escape mutants of each mAb:

53

EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T), and EsCAV/F11 (E144G). Thus, the

54

epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in

55

the VP1 structure, suggesting that VP1 has at least 2 different neutralizing epitopes. However,

56

MoCAV/F8 did not react to EsCAV/F2 or to EsCAV/F8, suggesting that binding of

57

MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In

58

addition, MoCAV/F2, with a titer of 1:12,800 to the parent strain, neutralized EsCAV/F2 and

59

EsCAV/F8 with low titers of 32 and 152, respectively. The similarity of the reactivity of

60

MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope

61

recognized by these mAbs.

62 63 64 65 2

66

INTRODUCTION

67

Chicken anemia virus (CAV), which was first isolated in Japan (Yuasa et al., 1979), is

68

known to be ubiquitous worldwide. CAV causes a disease characterized by severe anemia,

69

retarded growth, intramuscular and subcutaneous hemorrhages, lymphoid depletion, and

70

increased mortality in young chickens (Taniguchi et al., 1982; Yuasa et al., 1979).

71

CAV is a nonenveloped virus and is classified into the Gyrovirus genus of the family

72

Circoviridae (King et al., 2011). The viral genome consists of a negative-sense, circular,

73

single-stranded 2.3-kb DNA genome with 3 partially overlapping ORFs (ORF1, ORF2,

74

ORF3) encoding the proteins VP1 (52 kDa), VP2 (24 kDa), and VP3 (14 kDa), respectively.

75

VP1 is the only structural protein known to form the viral capsid (Todd et al., 1990).

76

Although the non-structural VP2 function is unknown, it has been proposed that VP2 may act

77

as a scaffold protein during virion assembly to facilitate the correct conformation of VP1

78

(Noteborn et al., 1998). The non-structural VP3 protein, also called apoptin, causes apoptosis

79

in chicken thymocytes and chicken lymphoblastoid T cell lines (Noteborn et al., 1994).

80

All CAV isolates belong to one serotype (McNulty, 1991; Yuasa & Imai, 1986), and the

81

amino acid (aa) composition of VP1 is considered to be highly conserved, although Renshaw

82

et al. (1996) reported a hypervariable region within VP1 (aa positions 139 to 151) and that aa

83

changes within this region influenced the rate of virus replication in cell cultures.

84

In a phylogenetic analysis of full-length deduced VP1 aa sequences, 3 distinct clusters

85

were reported, and a common signature aa profile (I/T75, L97, Q139, and Q144) could be

86

identified only for genetic group II (Ducatez et al., 2006; Islam et al., 2002). Another major

87

aa profile (V75, M97, K139, and E144) was also found (Hailemariam et al., 2008; van Santen

88

et al., 2001). However, there is a lack of information related to the antigenicity of CAV

89

strains belonging to these genetic groups. 3

90

There have been some reports on the production of monoclonal antibodies (mAbs) to

91

CAV, particularly focusing on those with neutralizing activity. McNulty et al. (1990) reported

92

that mAbs to a Cux-1 strain showed 3 staining types in infected cells in immunofluorescent

93

staining, and only 3 of 4 Type 1 mAbs had neutralizing activity. However, the epitopes

94

recognized by these mAbs were not analyzed. Immunofluorescent staining with the mAbs

95

indicated antigenic differences among the 5 CAV strains tested (McNulty et al., 1990). In

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another study, 8 mAbs were generated but lacked virus-neutralizing activity (Chandratilleke

97

et al., 1991). Recently, one VP1-specific mAb was established by immunization of mice with

98

truncated recombinant VP1; however, its virus-neutralizing activity was not evaluated (Lien

99

et al., 2012). Thus, the neutralizing epitopes of CAV remain poorly understood. Scott et al.

100

(1999) reported that most of the molecularly cloned viruses derived from Cux-1 after 310 cell

101

culture passages showed weak reactivity to the neutralizing 2A9 mAb (McNulty et al., 1990)

102

compared with the low-passaged cloned viruses, and the aa at position 89 in VP1 appeared to

103

be crucial for determining its reactivity with the mAb 2A9.

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MAbs could be very useful and powerful tools for understanding the pathogenesis,

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isolate characterization, and epidemiology, or for improving CAV diagnosis. In this paper, we

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describe the production and characterization of CAV mAbs and the expected epitopes

107

recognized by neutralizing mAbs. To the best of our knowledge, this is the first report on

108

epitope mapping of VP1 using neutralizing mAbs. Furthermore, we also genetically

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characterized two antigenic CAV groups that were differentiated by the mAbs.

110 111

RESULTS

112

Establishment of hybridomas secreting antibodies to CAV 4

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Four hybridomas secreting CAV antibodies were established from a mouse immunized

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with CAV, and were designated as MoCAV/F2 (IgG1), MoCAV/F8 (IgG1), MoCAV/F11

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(IgG2b), and MoCAV/E6 (IgG2a). Three of the mAbs (MoCAV/F2, MoCAV/F8, and

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MoCAV/F11) showed neutralizing activity at titers ranging from 1:12,800 to 1:25,600 (Table

117

1), but MoCAV/E6 did not.

118

The immunofluorescent staining patterns of the A2/76-infected MSB1 cells with the

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mAbs could be classified into 2 types when observed within 36 h post-infection (hpi), as

120

shown in Figs. 1a and 2. Diffused, irregularly shaped granular staining with MoCAV/F2,

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MoCAV/F8, and MoCAV/F11 was observed in the enlarged infected cells, whereas scattered,

122

spherically shaped antigens of various sizes were observed in the infected cells reacted with

123

MoCAV/E6.

124

Immunoprecipitation

showed

that

MoCAV/F2,

MoCAV/F8,

and

MoCAV/F11

125

precipitated a protein band of an estimated size of 50 kDa, corresponding to the VP1 (50

126

kDa) protein in the infected MSB1 cell lysates (Fig. 1c); however, MoCAV/E6 did not

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precipitate this protein and also failed to precipitate any other viral protein. An mAb to the

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nucleoprotein of influenza A virus was used as a control, and did not precipitate CAV proteins.

129

The VP1 recombinant proteins expressed in Cos7 cells using a pcDNA3.1 (+) vector

130

were reacted with neutralizing mAbs as well as anti-VP1 peptide antibodies (Fig. 1d), while

131

they were not with MoCAV/E6 (data not shown).

132 133

Viral protein expression in A2/76-infected MSB1 cells

5

134

The kinetics of the expression of viral antigens was examined using 3 neutralizing mAbs

135

and MoCAV/E6 in infected MSB1 cells fixed at different time points (6, 12, 24, 36, 48, 60,

136

and 72 hpi).

137

Although there was no fluorescent signal observed at 6 hpi (data not shown), positive

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immunofluorescece was observed with all of the mAbs at 12 hpi; however, only MoCAV/E6

139

showed clearer and stronger fluorescence compared with the other mAbs (Fig. 1a).

140

The irregular-shaped small granules detected by the 3 neutralizing mAbs became

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stronger and clearer at 24 hpi than at 12 hpi, and reached a maximal level at 36 hpi, when

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they were distributed all over the cells. However, the staining pattern markedly changed

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toward misshapen fluorescent staining of various sizes at 60 hpi (Fig. 1a) and 72 hpi (data not

144

shown), which was most likely due to a cytopathic effect. Many infected cells with

145

misshapen antigens seemed not to be intact. An anti-VP1 peptide antibody confirmed the

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presence of VP1 antigen in the infected cells.

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The intensity of fluorescent signals detected by MoCAV/E6 peaked at 24 hpi and 36 hpi.

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MoCAV/E6 did not change its fluorescent staining pattern (scattered, spherical structures of

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various sizes) during the observation period, although the number of infected cells reduced

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and the signal became weak at later time points of 60 hpi (Fig. 1a) and 72 hpi (data not

151

shown).

152 153

Co-staining of A2/76-infected MSB1 cells with mAbs

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Co-staining patterns of A2/76-infected MSB1 cells with MoCAV/E6 and neutralizing

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mAbs were analyzed with a confocal microscope. Antigens detected by MoCAV/F11 and

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MoCAV/E6 were localized in the nuclei of infected cells as indicated by DAPI 6

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counterstaining (Fig. 1b). The merged image of antigens detected by both mAbs indicated

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that the antigen signals seemed to partially overlap. The same results were obtained in

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combinations of MoCAV/E6 with other mAbs (data not shown).

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Blocking indirect immunofluorescent antibody test (IFAT)

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Bindings of fluorescein-conjugated MoCAV/F2 and MoCAV/F8 were mutually

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competitively blocked, whereas the F11 and E6 competitors did not block the binding of the

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conjugated MoCAV/F2 and MoCAV/F8 (Fig. 2). On the other hand, fluorescein-conjugated

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MoCAV/F11 and MoCAV/E6 were not blocked by any competitor, except for homologous

166

mAbs.

167 168

Reactivity of neutralizing mAbs to heterologous CAV strains in the virus-neutralizing

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test (VNT)

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MoCAV/F2 and MoCAV/F8 neutralized all of the CAV strains examined (Table 2). By

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contrast, MoCAV/F11 could not neutralize G3/78, G5/79, G6/79, NI/77, or HY/80. Thus, the

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CAV strains were antigenically divided into 2 distinct groups based on MoCAV/F11; mAb

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Group 1 included CAVs recognized by MoCAV/F11, and Group 2 included CAVs not

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recognized by this mAb.

175 176

Phylogenetic analysis

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Phylogenetic analysis of full-length deduced VP1 aa sequences of the 2 mAb antigenic

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group strains in comparison with other strains available in GenBank resulted in 3 distinct 7

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clusters (Fig. 3a). The aa profiles in VP1 sequences of CAV strains are shown in Fig. 3b.

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MAb Group 2 strains (G5/79, G6/79, NI/77, and HY/80) with the aa profile of I75, L97,

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Q139, Q144, which were not neutralized with MoCAV/F11, fell into cluster II, and they

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formed a single genetic group with TR20, Arg729, 704, CAV-E, and SMSC-1 with the same

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profile. Although the G3/78 strain had the same profile, it was not used in phylogenetic

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analysis since its complete sequence was not conclusively determined due to the appearance

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of double peaks in some positions. Only 6 of all the strains classified in cluster II had a

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different profile (I/T/V75, L/M97, N/Q139, H/Q144).

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MAb Group 1 strains (A2/76, CAE26P4, 82-2, AO/77, A1/76, G7/91, IBA/94, NI/92,

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G1/74, and KY/80) with the aa profile of V75, M97, K139, E144, which were neutralized by

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MoCAV/F11, fell into cluster I (V75, M97, K139, E/D/N144), and III (V75, M97, K139,

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E144).

191 192 193 194

Antigenic and genetic characterizations of escape mutants One escape mutant was selected for each of MoCAV/F2, MoCAV/F8, and MoCAV/F11, designated as EsCAV/F2, EsCAV/F8, and EsCAV/F11, respectively.

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In IFAT of the MSB1 cells infected with escape mutants, MoCAV/F11 reacted to both

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EsCAV/F2 and EsCAV/F8, while both MoCAV/F2 and MoCAV/F8 recognized only

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EsCAV/F11. Chicken polyclonal antibody against A2/76 reacted with all of the escape

198

mutants (Fig. 4).

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MoCAV/F8 and MoCAV/F11 did not neutralize their corresponding escape mutants,

200

EsCAV/F8 and EsCAV/F11 (Table 1). Moreover, MoCAV/F8 did not neutralize EsCAV/F2.

201

By contrast, MoCAV/F2 neutralized both EsCAV/F2 and EsCAV/F8 with low dilutions of 8

202

1:32 and 1:152, respectively. IFAT was also conducted using escape mutants (EsCAV/F2 and

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EsCAV/F8), and neither MoCAV/F2 nor MoCAV/F8 reacted to the mutants at dilutions as

204

low as 1:10 (data not shown).

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Comparison of the VP1 aa sequence of the parent virus (A2/76) with those of the 3

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escape mutants revealed the deletion of threonine (T) and alanine (A) at positions 89 and 90

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(T89+A90) in EsCAV/F2, a single aa change of isoleucine (I) to T at position 261 (I261T) in

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EsCAV/F8, and a glutamic acid (E) to glycine (G) change at position 144 (E144G) in

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EsCAV/F11 (Table 1). Additionally, the 3 escape mutants did not show any aa changes in

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VP2 and VP3 in comparison to the parent virus (data not shown).

211 212

The titer of EsCAV/F2 was reduced by 1.75 log compared with that of the parent A2/76 strain in MSB1 cells (Table 1).

213 214

DISCUSSION

215

Three mAbs (MoCAV/F2, MoCAV/F8, MoCAV/F11) possessed neutralizing activity

216

with A2/76 and were directed against VP1 in immunoprecipitation analysis, while

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MoCAV/E6 did not show neutralizing activity and did not immunoprecipitate any of the VPs

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(Fig. 1c). This result was confirmed using Cos7 cells with the VP1 recombinant proteins (Fig.

219

1d), while MoCAV/E6 did not react to the recombinant proteins (data not shown). Moreover,

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MoCAV/E6 showed a different staining pattern from the other mAbs as shown in Figs. 1a and

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2. A co-staining study showed partial co-localizations of the antigens detected by MoCAV/E6

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and VP1 neutralizing mAbs (Fig. 1b). However, the time-course staining pattern of

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MoCAV/E6 was different from that of VP1 mAbs (Fig. 1a). Thus, MoCAV/E6 seemed not to

9

224

recognize VP1, although further study should be performed to define the target protein of

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MoCAV/E6.

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Douglas et al. (1995) reported that VP1 antigens in infected MSB1 cells were not

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detected using a VP1 non-neutralizing mAb (1H1) until 30 hpi, although VP2 and VP3

228

antigens were detectable as early as 12 hpi. By contrast, the present study showed that VP1

229

neutralizing mAbs could detect antigens as early as 12 hpi, indicating that VP1 is produced

230

early (Fig. 1a). The reason for this discrepancy between studies is unclear. Although the same

231

MSB1 cells were used in both studies, the CAV strains (A2/76 and Cux-1) tested were

232

different. However, since the both strains do not appear to have different biological properties,

233

the mAbs used in the experiment might have affected the results. Our mAbs showed

234

neutralization activity, whereas the mAb 1H1 lacks this activity. In addition, anti-VP1 peptide

235

antibody results supported the early detection of VP1 antigens detected by the neutralizing

236

mAbs (Fig. 1a).

237

Although several mAbs against CAV or recombinant VP1 proteins have been developed

238

(Chandratilleke et al., 1991; Lien et al., 2012; McNulty et al., 1990), there is nonetheless a

239

lack of important information related to the antigenicity of VP1, especially with respect to

240

neutralizing epitopes. We identified the neutralizing epitopes on VP1. Blocking IFAT showed

241

that the binding of MoCAV/F2 and MoCAV/F8 to the A2/76-infected cells was blocked by

242

mutual competition between the mAbs (Fig. 2). Moreover, the 2 mutants (EsCAV/F2 and

243

EsCAV/F8) did not react with either MoCAV/F2 or MoCAV/F8 (Fig. 4). These results

244

suggested that these mAbs recognize the same epitope on VP1. However, VP1 aa analysis of

245

these mutants revealed two different mutations in the epitopes; the deletion of T89+A90 in

246

EsCAV/F2 VP1, and I261T in EsCAV/F8 VP1 (Table 1). Kaverin et al. (2002) revealed the

247

antigenic sites on the hemagglutinin molecule of the H5 subtype avian influenza virus (AIV) 10

248

that have distinct aa sequences but are topographically close in the three-dimensional

249

structure, and partially overlap in reaction with mAbs.

250

antigenic sites, including aas T89+A90 and I261, are topographically close in the VP1

251

structure.

Therefore, it is likely that the

252

Scott et al. (2001) reported a variant virus, P310 2A9-resist, that resists neutralization by

253

mAb 2A9 (McNulty et al., 1990), which was selected from a Cux-1 strain that had been

254

passaged 310 times in MSB1 cells. Fluorescent VP1 antigens were not detectable by the mAb

255

in the cells infected with P310 2A9-resist virus, even at low antibody dilutions (1:100),

256

whereas the low-passage virus produced positive staining at high dilutions (≥1:80,000). The

257

authors suggested that the aa change at position 89 of VP1 was a key determinant of mAb

258

2A9 reactivity, because P310 2A9-resist virus, which has A89 instead of T89, produced no

259

immunofluorescence (1:100). Although mAb 2A9 neutralized the P310 2A9-resist virus at

260

very low dilutions (1:5), whether the mAb could react to antigens at lower dilutions than

261

1:100 in IFAT was not evaluated. In this study, a similar phenomenon was observed (Table 1).

262

Although MoCAV/F2 did not neutralize EsCAV/F2 at low dilutions (1:100), unexpectedly, it

263

was neutralized at even lower dilutions (1:32). Thus, these results suggest that the aa change

264

of T89A in VP1 is not necessarily a key determinant of MoCAV/F2 reactivity, since

265

EsCAV/F2 lacks the aas T89+A90.

266

These results raise questions as to why MoCAV/F2 could neutralize EsCAV/F2 at a high

267

mAb concentration (Table 1). One possible explanation for this phenomenon may be that the

268

epitope was not mutated completely; therefore, the corresponding mAb was still able to bind

269

to it but with weaker affinity. However, MoCAV/F2 also neutralized EsCAV/F8, which

270

possesses T89+A90, at a high mAb concentration. Although the reason for this phenomenon

271

is unclear, the results suggest that complete binding of MoCAV/F2 to the epitope might 11

272

require the coexistence of an antigenic site including I261, which is recognized by

273

MoCAV/F8. On the other hand, we could not explain why MoCAV/F2 did not recognize the

274

antigens in the infected cells with EsCAV/F2 even when using very low dilutions (e.g., 1:10),

275

which could be due to the low sensitivity of IFAT.

276

MoCAV/F8 could not only neutralize EsCAV/F8 but also EsCAV/F2 (Table 1). This

277

unexpected phenomenon may indicate that the binding of MoCAV/F8 to the epitope requires

278

coexistence of the epitope recognized by MoCAV/F2.

279

Kaverin et al. (2007) reported that some mAbs to the hemagglutinin molecule of the H5

280

subtype AIV have the ability to overlap 2 distinct antigenic sites. Similarly, MoCAV/F2 and

281

MoCAV/F8 might overlap 2 different antigenic sites that are close to each other in the three-

282

dimensional VP1 structure; therefore, any change at the mAb-binding site on the epitopes

283

could result in dramatic reduction or loss of mAb reactivity. However, the possibility of a

284

single epitope recognized by these mAbs cannot be denied considering the similar reactivity

285

of these VP1 mAbs, although the escape mutants of MoCAV/F2 and MoCAV/F8 displayed

286

different aa mutations in VP1. In this study, only one escape mutant for each neutralizing

287

mAb was examined. More escape mutants should be examined to obtain further information

288

about antigenic epitopes in VP1.

289

Wang et al. (2009) reported that the structure protein VP1 genes had undergone positive

290

selection, and 8 (75, 125, 139, 141, 144, 287, 370, 447) positively selected aa sites were

291

identified. In this study, only EsCAV/F11 showed an aa change corresponding to one of the

292

positively selected aa sites (E144G). Thus, it is likely that the escape mutants were selected

293

by antibody selection pressure, with the exception of EsCAV/F11.

294

Renshaw et al. (1996) indicated that one or both of the aa differences at positions 139

295

and 144 affected the rate of replication or the spread of infection in MSB1 sublines cells. In 12

296

this study, the aa change E144G did not affect the CAV replication rate in MSB1 cells (Table

297

1). Therefore, both of the aa changes at positions 139 and 144 might be required to affect the

298

CAV replication in MSB1 cells.

299

The CAV strains examined were phylogenetically grouped into 3 clusters (Fig. 3ab). In

300

cluster II, corresponding to group II described by Islam et al. (2002), 26 of the 32 strains had

301

the aa profile I75, L97, Q139, Q144, including all strains of mAb Group 2 that were not

302

neutralized by MoCAV/F11. A2/76 (V75, M97, K139, E144 profile) lost the neutralizing

303

epitope recognized by MoCAV/F11 owing to the aa change E144G (Table 1). However, this

304

change was not observed in the mAb Group 2 strains (I75, L97, Q139, Q144 profile), which

305

naturally lack the epitope recognized by MoCAV/F11. MoCAV/F11 reacted to the HK1/13

306

strain (V75, L97, N139, Q144 profile, cluster II) in IFAT. Thus, the aa change at position 144

307

is not necessarily associated with the loss of binding ability to MoCAV/F11. However, further

308

studies are required to determine whether all CAV strains (I75, L97, Q139, Q144 profile)

309

naturally lack the antigenic site(s) recognized by MoCAV/F11.

310

In conclusion, we established 4 mAbs, 3 of which (MoCAV/F2, MoCAV/F8, and

311

MoCAV/F11) had neutralizing activity and recognized the VP1 protein. Analysis of the

312

escape mutants of the neutralizing mAbs revealed at least 2 neutralizing epitopes on the VP1

313

protein, which have not been reported previously, to our knowledge. However, as the

314

reactivity of MoCAV/F2 and MoCAV/F8 to VP1 was similar, the existence of a single epitope

315

recognized by these mAbs cannot be denied. The CAV strains evaluated could be

316

differentiated into 2 distinct antigenic groups by MoCAV/F11, which could be associated

317

with specific aa profiles of VP1. Mutations in VP1 are known to affect pathogenicity in

318

chickens or viral replication in cells. However, there is no consistent molecular biological

13

319

evidence to explain the events, and there are still many aspects that remain unresolved with

320

respect to CAV biology.

321 322

METHODS

323

Cell culture

324

MDCC-MSB1 cells (MSB1 cells) were cultured in growth medium (GM) consisting of

325

RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% FBS and

326

10% Daigo’s GF 21 growth factor (Wako Junyaku) in a humidified incubator with 5% CO2 at

327

39.5°C.

328 329

Virus

330

The following CAV strains were used: A1/76, A2/76, AO/77, G1/74, G3/78, G5/79,

331

G6/79, KY/80, and NI/77 (Yuasa & Imai, 1986); HY/80, G7/91, IBA/94, NI/92, and HK1/13,

332

which were isolated from diseased chicks infected with CAV (unpublished data); CAA82-2

333

(Otaki et al., 1987); and 26P4, which was obtained from a vaccine (Intervet). CAV was

334

propagated in MSB1 cells (Yuasa, 1983).

335

Viral titers were determined as described by Imai and Yuasa (1990). Briefly, 20 µl of a

336

10-fold virus dilution was added to wells of a 96-well microplate containing 200 µl MSB1

337

cells (2 × 105 cells ml-1). Four wells were used for each dilution. The inoculated cells were

338

passaged every 3 days, in which 40 µl of the cell suspension was transferred to a new well

339

including 200 µl of GM. The negative wells were determined after 8 passages. The cultures

340

showing red color (no cell growth) due to a cytopathic effect were regarded as CAV-positive

341

(Yuasa, 1983). Virus titers were quantified as the TCID50 by the Behrens-Kärber method 14

342

(Behrens & Kärber, 1934).

343 344

Mouse immunization and mAb production

345

A2/76 propagated in MSB1 cells was partially purified and concentrated as described

346

previously (Imai et al., 1991), and then used as the inoculum in 4 BALB/c mice. The titer of

347

the inoculum was approximately 1010 TCID50 ml-1. Each mouse was immunized with 3

348

intraperitoneal injections of 0.1 ml of the inoculum emulsified in Freund’s adjuvant every 3

349

to 4 weeks. One mouse showing the highest immunofluorescent titer was intravenously

350

injected with 0.1 ml of the inoculum. Four days later, spleen cells were fused with

351

P3X63Ag8U.1 myeloma cells in the presence of polyethylene glycol, and the fused cells

352

were selected and cultivated in GM supplemented with hypoxanthine, aminopterin, thymidine,

353

endothelial cell growth supplement (Becton Dickinson), and insulin-transferrin-selenium-S

354

supplement (Life Technologies) according to standard procedures. Antibody-positive

355

hybridoma cells were selected by IFAT (described below) and cloned 2 or 3 times by limiting

356

dilution.

357

Ascites was obtained by intraperitoneal injection with approximately 107 hybridoma

358

cells into a BALB/c mouse that had been primed with incomplete Freund’s adjuvant, as

359

described previously (Harlow & Lane, 1988). Isotypes of mAbs were determined in an

360

ELISA using a commercial kit (mouse monoclonal antibody isotyping reagents; Sigma-

361

Aldrich).

362

All mouse studies were conducted in compliance with the institutional rules for the care

363

and use of laboratory animals, and using protocols approved by the relevant committee at the

364

institution.

365 15

366

Immunoprecipitation

367

An IgG fraction of the ascites including mAb was precipitated by 33% saturated

368

ammonium sulfate and dialyzed with PBS. Protein concentration of the semi-purified IgG

369

mAb was determined using the Lowry method (Lowry et al., 1951).

370

A2/76-infected and uninfected MSB1 cells (negative control) were harvested at 48 hpi.

371

Immunoprecipitation was conducted according to the instructions of a commercial kit

372

(Immunoprecipitation Kit, Roche). Briefly, the cells (107 cells ml-1) were lysed in 50 mM

373

Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% nonidet P40, and 0.5% sodium

374

deoxycholate. The lysed cells were labeled with biotin-7-NHS (EZ-LinkTM Sulfo-NHS-LC-

375

Biotinylation Kit, Thermo), according to manufacturer instructions. The lysates were

376

precleaned with Protein G-agarose and mouse IgG-agarose (Sigma-Aldrich) followed by

377

incubation with mAb for at least 3 h at 4°C. Then 50 µl of Protein G-agarose was added to

378

the mixture and incubated for at least 3 h at 4°C. Fifty microliters of gel loading buffer (0.06

379

M Tris-HCl, pH 6.8; 10% (w/v) glycerol; 2% (w/v) SDS; 0.005% (w/v) bromophenol blue)

380

was added per complex after washing. The sample was boiled for 5 min and quenched on ice.

381

The mAb to influenza A virus nucleoprotein (Serotec Ltd.) was used as a negative control.

382

The samples obtained in immunoprecipitation were analyzed using 17% low bis-

383

polyacrylamide slab gels according to Hirano (1989). Transfer of the proteins from the gel to

384

a nitrocellulose membrane (0.22-µm pore size; Bio-Rad Laboratories) was conducted using a

385

semi-dry apparatus. Nonspecific binding sites on the membrane were blocked by incubation

386

with 3% BSA in PBS.

387

Biotin-labeled viral proteins were detected by a streptavidin-horseradish conjugate

388

(Sigma-Aldrich), and visualized with a chemiluminescent substrate using LAS-3000 (Fuji 16

389

Film). A molecular-weight standard (Precision Plus ProteinTM WesternCTM Standards; Bio-

390

Rad Laboratories) was incubated with Precision StreptTactin-HRP conjugate (Bio-Rad

391

Laboratories) and visualized as described above.

392

393

Expression of VP1 recombinant protein

394

The full coding gene of the VP1 protein was amplified using the following primers:

395

CAV-VP1-F EcoRI: 5’ −GCGGAATTCATGGCAAGACGAGCTCGCAGA−3’ and CAV-

396

VP1-R XhoI 5’−AATCTCGAG TCAGGGCTGCGTCCCCCAGTA−3’. The PCR product

397

was digested with restriction enzymes, EcoRI and XhoI (Invitrogen) and purified using a

398

GeneClean II Kit (MP Biomedicals). The purified VP1 gene was ligated into a pcDNA3.1 (+)

399

plasmid vector (Invitrogen) using a DNA ligation kit (Takara) according to the manufacture’s

400

instruction, and transformed into DH5α competent cells. After culture overnight at 37°C,

401

plasmid DNA was extracted by a Miniprep kit (QIAgene). The constructed recombinant

402

plasmid, pcDNA3.1 (+)−VP1, was characterized by restriction enzymes analysis (EcoRI and

403

XhoI) and sequencing, and then was purified using a EndoFree Plasmid Kit (QIAgene).

404

To express the VP1 protein in mamalian cells, 0.3 µg of pcDNA3.1 (+)−VP1 plasmids

405

per well were transfected into Cos7 cells cultivated in a Lab-Tek® Chamber Slide (NUNC)

406

by TransIT-LT1 Transfection Reagent (Mirus Bio) according to the manufacture’s instruction.

407

Mock cells were transfected with pcDNA3.1 (+) plasmids. At 36 h post transfection, the cells

408

were fixed with acetone for 10 min and were used to react with mAbs in IFAT (described

409

below).

410

17

411

IFAT

412

An IFAT using MSB1 cells was performed to detect CAV antigens or antibodies

413

according to the method of Yuasa et al. (1985). Briefly, CAV-infected cells were smeared

414

onto a glass microscope slide, dried, and fixed with acetone for 10 min. The antigen slides

415

were incubated with the culture supernatant of hybridoma cells, CAV mAb at approximately

416

3 µg ml-1, rabbit antiserum to VP1 peptide (1:200), or chicken antiserum to A2/76 (1:40), and

417

then with FITC-conjugated rabbit anti-mouse IgG (Rockland), goat anti-rabbit IgG (Sigma-

418

Aldrich), or rabbit anti-chicken IgG (Rockland) at 37°C for 30 min after washing with PBS

419

(pH 7.4). In some experiments, a low dilution (1:10) of mAbs (MoCAV/F2 and MoCAV/F8)

420

was used. Anti-VP1 peptide serum was prepared by immunizing a rabbit with a specific

421

peptide (CWDVNWANSTMYWESQ; Qiagen). The fluorescent signal was observed under a

422

fluorescence microscope (Biorevo BZ-9000, Keyence). The A2/76-infected cells were

423

prepared on a glass microscope slide at 6, 12, 24, 36, 48, 60, and 72 hpi following infection,

424

as described above. The antigen slides were then used for IFAT with the mAbs to examine the

425

kinetics of viral antigen expression.

426

Co-staining of A2/76-infected cells with mAbs was conducted to examine the

427

localization of antigens. Briefly, the antigen slides were incubated with a combination of each

428

of 2 mAbs as primary antibodies at 37°C for 30 min, and then with a combination of 2

429

isotype-conjugates [rabbit anti-mouse IgG1-Rhodamine (Rockland), goat anti-mouse IgG2a-

430

FITC (Southern Biotechnology), or goat anti-mouse IgG2b-Rhodamine (Santa Cruz

431

Biotechnology)] after washing with PBS. DAPI (Sigma-Aldrich) was used to counterstain the

432

cell nuclei. The localization of antigens detected by mAbs was analyzed using a confocal

433

microscope (Leica Microsystems).

434 18

435

Blocking IFAT

436

The A2/76-infected MSB1 cells prepared as described above were reacted with mAbs

437

at 5 µg ml-1 (MoCAV/F2, F8, or F11) or 200 µg ml-1 (MoCAV/E6) for 30 min at 37°C. After

438

washing with PBS, mAbs that were labeled with isotype fluorescence using a Zenon® mouse

439

IgG labeling kit (Life Technologies) were reacted for 30 min. After washing, the fluorescent

440

signal was observed under the fluorescence microscope.

441 442

VNT

443

A VNT was performed according to the microtest method of Imai and Yuasa (1990).

444

Briefly, in the α-neutralization procedure (constant-mAb, diluted-virus), 10-fold stepwise

445

dilutions of CAV were mixed with mAb (1:100) or GM (virus control), and then the mixtures

446

were incubated overnight at 4°C. Afterward, 20 µl of each mixture was inoculated to each of

447

4 wells with 200 µl of MSB1 cells (2 × 105 cells ml-1). The inoculated cells were passaged

448

every 3 days. The virus titer of the mixture was determined as described above, and the

449

neutralizing index was calculated based on the differences of virus titers (log10 TCID50)

450

between the mixtures with mAb and the virus control.

451

In the β- neutralization procedure (constant-virus, diluted-mAb), serial 2-fold dilutions

452

of mAb, beginning with a 1:100 dilution for the A2/76 strain or with a 1:2 dilution for the

453

escape mutants, were mixed with an equal amount of CAV containing 200 TCID50 0.1 ml-1.

454

The mixture was incubated overnight at 4°C and then inoculated into the wells containing

455

cells, followed by cell passaging as described above. Endpoint titers corresponding to 50%

456

neutralization were calculated by the Behrens-Kärber method. The reciprocal of the highest

457

dilution of mAb neutralizing 50% of CAV was taken as the antibody titer. 19

458 459

Selection of escape mutants

460

The undiluted virus stock of the A2/76 strain (approximately 107 TCID50 ml-1) was

461

mixed with mAbs (1:10). Original antibody titers of the 3 mAbs used are shown in Table 1.

462

After incubation for 1.5 h at 37°C followed by overnight incubation at 4°C, the mixture was

463

inoculated into 7 test tubes containing MSB1 cells (2 × 105 cells ml-1), and the inoculated

464

cells were passaged every 3 days up to 8 times. The viruses that were not neutralized,

465

indicated by the red color of the culture, were cloned 2 or 3 times by limiting dilutions using

466

MSB1 cells.

467 468 469 470

DNA extraction and PCR Viral DNA was extracted from CAV-infected MSB1 cell culture fluids using a QIAamp DNA blood Mini kit (QIAGEN).

471

Primers used for sequencing of full-length CAV VP1 genes were selected based on the

472

Cux-1 sequence (accession no. M55918, Noteborn et al. (1991)) and are available upon

473

request. The PCR protocol was conducted as described previously (Imai et al., 1998) except

474

that 50°C was used as the annealing temperature.

475 476

Sequencing and phylogenetic analysis

477

VP1, VP2, and VP3 genes of escape mutants of A2/76 selected by mAbs, and VP1 gene

478

sequences of CAV strains used in this study, except A2/76, G6/79, 26P4, and CAA 82-2, were

479

determined by direct sequencing using a BigDye Terminator v3.1 cycle sequencing kit 20

480

according to the manufacturer’s instructions (Life Technologies). Nucleotide sequencing was

481

performed using an Applied Biosystems 3500 Genetic Analyzer (Life Technologies). VP1

482

gene sequences of A2/76, G6/79, 26P4, and CAA 82-2, and VP2 and VP3 genes of A2/76

483

were obtained from GenBank.

484

Nucleotide sequences obtained were analyzed using GENETYX ver. 10 software

485

(GENETYX Corp.) and compared with other available sequences using the BLAST program.

486

The nucleotides and translated aa sequences were aligned with Clustal W (Thompson et al.,

487

1994). A phylogenetic tree of the VP1 gene was constructed using the maximum likelihood

488

method based on the Poisson correction model, supported by 500 bootstrap replicates. The

489

tree was drawn to scale, with branch lengths corresponding to the number of substitutions per

490

site. All positions containing alignment gaps and missing data were eliminated in complete

491

deletion (complete deletion option). Evolutionary analyses were conducted in MEGA 5

492

software (Tamura et al., 2011).

493 494

ACKNOWLEDGMENTS

495

We would like to thank Dr. Noboru Yuasa for critical reading of this manuscript and

496

helpful suggestions. We would also like to thank Sachiko Matsuda for the excellent technical

497

assistance.

498 499

REFERENCES

500 501 502 503 504

Behrens, B. & Kärber, G. (1934). Wie sind Reihenversuche für biologishe Auswertungen am zweckmäßigsten anzuordnen [in German]? Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 18, 379-388. Chandratilleke, D., O'Connell, P. & Schat, K. A. (1991). Characterization of proteins of chicken infectious anemia virus with monoclonal antibodies. Avian Dis 35, 854-862. 21

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Douglas, A. J., Phenix, K., Mawhinney, K. A., Todd, D., Mackie, D. P. & Curran, W. L. (1995). Identification of a 24 kDa protein expressed by chicken anaemia virus. J Gen Virol 76 ( Pt 7), 1557-1562. Ducatez, M. F., Owoade, A. A., Abiola, J. O. & Muller, C. P. (2006). Molecular epidemiology of chicken anemia virus in Nigeria. Arch Virol 151, 97-111. Hailemariam, Z., Omar, A. R., Hair-Bejo, M. & Giap, T. C. (2008). Detection and characterization of chicken anemia virus from commercial broiler breeder chickens. Virol J 5, 128. Harlow, E. & Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 274-275. Hirano, H. (1989). Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel. J Protein Chem 8, 115-130. Imai, K., Maeda, M. & Yuasa, N. (1991). Immunoelectron microscopy of chicken anemia agent. J Vet Med Sci 53, 1065-1067. Imai, K., Mase, M., Yamaguchi, S., Yuasa, N. & Nakamura, K. (1998). Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction. Res Vet Sci 64, 205-208. Imai, K. & Yuasa, N. (1990). Development of a microtest method for serological and virological examinations of chicken anemia agent. Nihon Juigaku Zasshi 52, 873-875. Islam, M. R., Johne, R., Raue, R., Todd, D. & Muller, H. (2002). Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences. J Vet Med B Infect Dis Vet Public Health 49, 332-337. Kaverin, N. V., Rudneva, I. A., Ilyushina, N. A., Varich, N. L., Lipatov, A. S., Smirnov, Y. A., Govorkova, E. A., Gitelman, A. K., Lvov, D. K. & Webster, R. G. (2002). Structure of antigenic sites on the haemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants. J Gen Virol 83, 2497-2505. Kaverin, N.V., Rudneva, I.A., Govorkova, E.A., Timofeeva, T.A., Shilov, A.A., Kochergin-Nikitsky, K.S., Krylov, P.S. & Webster, R.G. (2007). Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies. J Virol 81, 12911-12917. King., A. M. Q., Lefkowitz., E., Adams., M. J. & Carstens., E. B. (2011). Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses: Elsevier. Lien, Y. Y., Huang, C. H., Sun, F. C., Sheu, S. C., Lu, T. C., Lee, M. S., Hsueh, S. C., Chen, H. J. & Lee, M. S. (2012). Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus. J Vet Sci 13, 73-79. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-275. McNulty, M. S. (1991). Chicken anaemia agent: a review. Avian Pathol 20, 187-203. McNulty, M. S., Mackie, D. P., Pollock, D. A., McNair, J., Todd, D., Mawhinney, K. A., Connor, T. J. & McNeilly, F. (1990). Production and preliminary characterization of monoclonal antibodies to chicken anemia agent. Avian Dis 34, 352-358. Noteborn, M. H., de Boer, G. F., van Roozelaar, D. J., Karreman, C., Kranenburg, O., Vos, J. G., Jeurissen, S. H., Hoeben, R. C., Zantema, A., Koch, G. & et al. (1991). Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. J Virol 65, 3131-3139. 22

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Noteborn, M. H., Todd, D., Verschueren, C. A., de Gauw, H. W., Curran, W. L., Veldkamp, S., Douglas, A. J., McNulty, M. S., van der, E. A. & Koch, G. (1994). A single chicken anemia virus protein induces apoptosis. J Virol 68, 346-351. Noteborn, M. H., Verschueren, C. A., Koch, G. & Van der Eb, A. J. (1998). Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope. J Gen Virol 79 ( Pt 12), 3073-3077. Otaki, Y., Nunoya, T., Tajima, M., Tamada, H. & Nomura, Y. (1987). Isolation of chicken anaemia agent and Marek's disease virus from chickens vaccinated with turkey herpesvirus and lesions induced in chicks by inoculating both agents. Avian Pathol 16, 291-306. Renshaw, R. W., Soine, C., Weinkle, T., O'Connell, P. H., Ohashi, K., Watson, S., Lucio, B., Harrington, S. & Schat, K. A. (1996). A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 70, 8872-8878. Scott, A. N., Connor, T. J., Creelan, J. L., McNulty, M. S. & Todd, D. (1999). Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages. Arch Virol 144, 1961-1975. Scott, A. N., McNulty, M. S. & Todd, D. (2001). Characterisation of a chicken anaemia virus variant population that resists neutralisation with a group-specific monoclonal antibody. Arch Virol 146, 713-728. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28, 2731-2739. Taniguchi, T., Yuasa, N., Maeda, M. & Horiuchi, T. (1982). Hematopathological changes in dead and moribund chicks induced by chicken anemia agent. Natl Inst Anim Health Q (Tokyo) 22, 61-69. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680. Todd, D., Creelan, J. L., Mackie, D. P., Rixon, F. & McNulty, M. S. (1990). Purification and biochemical characterization of chicken anaemia agent. J Gen Virol 71 ( Pt 4), 819-823. van Santen, V. L., Li, L., Hoerr, F. J. & Lauerman, L. H. (2001). Genetic characterization of chicken anemia virus from commercial broiler chickens in Alabama. Avian Dis 45, 373-388. Wang, D., Fan, W., Han, G. Z. & He, C. Q. (2009). The selection pressure analysis of chicken anemia virus structural protein gene VP1. Virus Genes 38, 259-262. Yuasa, N. (1983). Propagation and infectivity titration of the Gifu-1 strain of chicken anemia agent in a cell line (MDCC-MSB1) derived from Marek's disease lymphoma. Natl Inst Anim Health Q (Tokyo) 23, 13-20. Yuasa, N. & Imai, K. (1986). Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (CAA). Avian Pathol 15, 639-645. Yuasa, N., Taniguchi, T. & Yoshida, I. (1979). Isolation and Some Characteristics of an Agent Inducing Anemia in Chicks. Avian Dis 23, 366-385. 23

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Yuasa, N., Imai, K. & Tezuka, H. (1985). Survey of antibody against chicken anaemia agent (CAA) by an indirect immunofluorescent antibody technique in breeder flocks in Japan. Avian Pathol 14, 521-530.

24

604 Table 1. Characterization of escape mutants Titer of virus Escape mutant

Amino acid change

(TCID50 ml-1)

Neutralizing antibody titers of mAb to escape mutants* F2

F8

F11

32

3.5

2.5

>3.5

4.0

3.5

>5.0 >3.5

2.5

>4.0

4.5

4.0

F11

>5.0

5.5

3.5

>4.0

>4.0

>5.5

3.0

>4.0

>6.0

0.0

0.0

0.5

0.0

0.5

NI /77

HY /80 4.0

Virus neutralization test was performed using the α-neutralization procedure described in the Materials and Methods.

619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 26

635

Figure Legends

636

Fig. 1. (a) Viral protein expression kinetics in CAV A2/76-infected MSB1 cells.

637

Immunofluorescent antibody tests were conducted to detect antigens using mAbs

638

(MoCAV/F2, F8, F11, E6) and a rabbit serum anti-VP1 peptide. Mouse normal ascitic fluid

639

was used as a negative control. Infected cells were collected at 6, 12, 24, 36, 60, and 72 hpi

640

and used as antigens. Scale bar = 10 µm.

641

(b) Co-staining of A2/76-infected MSB1 cells with mAbs. The infected cells were stained

642

with a neutralizing mAb (MoCAV/F11) and a non-neutralizing mAb (MoCAV/E6), and then

643

with IgG subclass-specific secondary antibodies labeled with rhodamine for MoCAV/F11,

644

and with FITC for MoCAV/E6. Infected cells collected at 36 hpi were used as antigens. Cell

645

nuclei were counterstained with DAPI. The fluorescent signals were observed under the

646

confocal microscope.

647

(c) Immunoprecipitation analysis of A2/76-infected MSB1 cells. The infected or uninfected

648

cell lysates collected at 48 hpi were biotin-labeled and immunoprecipitated with mAbs

649

against CAV and against influenza A virus nucleoprotein (NP). The immunoprecipitated

650

samples were analyzed by SDS-PAGE, and then the biotin-labeled proteins were transferred

651

from a gel to a nitrocellulose membrane. Biotin-labeled viral proteins were detected by a

652

streptavidin-horseradish conjugate and visualized with the chemiluminescent substrate. M:

653

molecular-weight standard. F2 (MoCAV/F2), F8 (MoCAV/F8), F11 (MoCAV/F11), E6

654

(MoCAV/E6), and NP (negative control) were used to immunoprecipitate viral proteins in

655

infected cells; F2 MSB1, F8 MSB1, F11 MSB1, E6 MSB1, and NP MSB1 indicate the

656

treatment of uninfected cells with mAbs described above.

657

(d) Reactivity of neutralizing mAbs with recombinant VP1 proteins expressed in Cos7 cells.

658

The full-length of the VP1 gene was cloned into pcDNA3.1 (+) vector. The constructed 27

659

pcDNA3.1 (+)-VP1 plasmids were transfected into Cos7 cells. IFAT were conducted using

660

the VP1 expressed cells with mAbs (MoCAV/F2, F8, F11) and anti-VP1 peptide antibody.

661

Mouse normal ascitic fluid was used as a negative control. Mock cells that were transfected

662

with pcDNA3.1 (+) vector were also used as negative control. Scale bar = 10 µm.

663 664

Fig. 2. Blocking immunofluorescent antibody tests. Blocking tests were conducted using

665

mAbs (MoCAV/F2, F8, F11, or E6) as competitors and mAbs directly labeled with

666

fluorescein. Infected cells collected at 36 hpi were used as antigens. Scale bar = 10 µm.

667 668

Fig. 3. (a). Phylogenic analysis of the complete deduced amino acid sequences of the CAV

669

VP1 protein. (▲): G1/74, KY/80, AO/77, 26P4, A2/76, A1/76, CAA 82-2, G7/91, IBA/94,

670

and NI/92; (■): G5/79, G6/79, NI/77, and HY/80. Sequences from GenBank are indicated

671

with the country name followed by accession number. The phylogenetic tree was constructed

672

using the maximum likelihood method based on the Poisson correction model for amino

673

acids in MEGA5 software, supported by 500 bootstrap replicates. The scale bar shows the

674

number of base substitutions per site.

675

(b) Comparison of the amino acid residues of VP1 of CAV strains in clusters I, II, and III.

676

Alignment was conducted with Clustal W. Numbering (70–150) was based on the Cux-1 VP1

677

sequence (accession no. M55918, Noteborn et al., 1991). Sequences of strains used in

678

neutralization tests are underlined. The amino acids at positions 75, 97, 139, and 144 are

679

boxed.

680

Fig. 4. Reactivity of mAbs in MSB1 cells infected with escape mutants. Immunofluorescent

681

antibody tests were conducted using neutralizing mAbs (MoCAV/F2, F8, and F11), and 28

682

chicken anti-A2/76 serum to detect antigens in the cells infected with escape mutants

683

collected at 36 hpi. Scale bar = 10 µm.

684

29

Figure 1a Click here to download Figure: Fig.1a.JGV-D-14-00081.pptx

Fig. 1a

12 hpi

MoCAV/F2

MoCAV/F8

MoCAV/F11

MoCAV/E6

Anti-VP1 peptide

24 hpi

36 hpi

60 hpi

Figure 1b Click here to download Figure: Fig. 1bJGV-D-14-00081.pptx

Fig. 1b

MoCAV/F11

Merge

MoCAV/E6

DAPI

Figure 1c Click here to download Figure: Fig. 1c VIR.2014.065789.pptx

Fig. 1c

F11

kDa

75 -

50 -

F11

MSB1

E6 E6 MSB1

NP

M

NP

MSB1

F8 F8

MSB1

F2

kDa 75 50 -

37 37 -

M

F2

MSB1

Figure 1d Click here to download Figure: Fig 1d.JGV-D-14-00081.pptx

MoCAV/F2

VP1expressed cells

Mock cells

MoCAV/F8

MoCAV/F11

Anti-VP1 peptide

Normal Ascitic fluid

Figure 2 Click here to download Figure: Fig. 2-IFAT VIR.2014.065789.pptx

Fig. 2

MoCAV/F2

Competitor mAbs

MoCAV/F2

MoCAV/F8

MoCAV/F11

MoCAV/E6

Fluorescein-labeled mAbs MoCAV/F8

MoCAV/F11

MoCAV/E6

Figure 3a Click here to download Figure: Fig. 3a VIR.2014.065789.pptx

Fig. 3a

Fig. 3b

Figure 3b ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| Click here to download Figure: Fig. 3b VIR.2014.065789.pdf 80 90 100 110 120 130 140 150 AB119448.1/G6 EU871778.1/Arg729 U65414.1/704 AB027470.1/TR20 AY583757.1/CAV-E KJ004669/NI/77 KJ004668/HY/80 KJ004667/G5/79 AF285882.1/SMSC-1 HQ872024.1/JS-China HQ872026.1/HU-China HQ872027.1/HU-China 6 HQ872043.1/JS-China 66 EU871782.1/Arg753 AY839944.2/LF4 KJ126838/HK1/13 HQ872045/JS-China 6 DQ124936.1/AH4 KC691410.1/CMB12077 KC691411.1/CMB11090 AJ888521.1/116 AF395114.1/BD-3 AJ888524.1/118 DQ016140.1/69 U69549.1/L-028 AF311900.3/98D06073 HQ872031.1/AN-China 36 L14767.1/CIA-1 FR850026.1/JS-China 85 HQ872029.1/JS-China 22 AY583758.1/CAV-P FJ883782.1/INDAP07 M55918.1/CAECUX1 JF712621.1/CAV-India U69548.1/ConnB FJ896818.1/INDKA06 M81223.1/Cux1 U66304.1/CAU66304 AF390038.1/3-1 KM226339/G1/74 KM226341/KY/80 AF475908.1/Harbin KM226338/AO/77 D10068.1/CAE26P4 AF527037.1/BL-5 AF227982.1/CAU269/7 AY150576.1/BL-5P90 AB031296.1/A2 AF311892.2/98D02152 EF683159.1/3711 AF313470.1/Del-Ros KM226337/A1/76 S71488.1/Pallister AY843527.2/TJBD33 D31965.1/82-2 AB046590.1/AH9410 HQ872025.1/HU-China 13 KM226342/G7/91 KM226340/IBA/94 KM226343/NI/92

RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIKFLTE RFQGIKFLTE RFQGIKFLTE RFQGIIFLTE RFQGIIFLTE RFQGVIFLTE RFQGVIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGTIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGIIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE RFQGVIFLTE

GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA GLILPKNSTA

GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GEYADHLYGA GSYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHMYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHLYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GDYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMFGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA GGYADHMYGA

RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK RVAKISVNLK

EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLVSMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT EFLLASMNLT

YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKLGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKIGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA YVSKLGGPIA

GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSNP GELIADGSNP GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSQS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKA GELIADGSKS GELIADGSKS GELIADGSKS GELIADGSKS

QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAQNWPNCW QAAHNWPNCW QAAHNWPNCW QAADNWPNCW QAADNWPNCW QAGNNWPNGC QAADNWPNCW QAADNWPNCW LAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAEENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAKNWPNCW EAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW EAAENWPNCC QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW QAAENWPNCW

Cluster II

Cluster I

Cluster III

Figure 4 Click here to download Figure: Fig. 4 VIR.2014.065789.pptx

Fig. 4

MoCAV/F2

EsCAV/F2

EsCAV/F8

EsCAV/F11

MoCAV/F8

MoCAV/F11

Chicken anti A2/76 serum